An immune system, generally initiated through an innate immune system, should be elaborately controlled to keep its balance. That is, the balances between immunity and tolerance, between T helper type 1 (Th1) and T helper type 2 (Th2) immunities, and between inflammation and unresponsiveness should be necessarily controlled elaborately. Unfortunately, conditions such as autoimmune-related diseases, allergic diseases, chronic inflammation, etc. have, however, been spread since many therapeutic agents for the immune-related diseases developed up to now could not control the immune system adequately. However, the innate immune system is a mechanism in which immune cells are activated by recognizing a structural difference of a foreign substance (Pathogen-Associated Molecular Pattern, PAMP) when a pathogen was invaded, and subsequent signals are transmitted to initiate a cascade reaction of the immune system, resulting in destruction of the pathogen. Accordingly, therapeutic agents of the immune-related diseases should be necessarily developed to minimize the Evil mechanism after exact understanding of a Good & Evil mechanism using the innate immune system.
In the 1890's, William B. Coley observed a surprising result that infection of pathogenic microorganisms may induce an anti-cancer effect in cancer patients, and therefore it was found that its modified bacterial therapy has about 40% of the therapeutic effect if it is subject to 900 cancer patients. In the 1980's, Japanese researchers recognized utility of Coley's toxin in a different and new aspect, proved that an active fraction of Bacillus Calmette-Guerin (BCG) shows an anti-cancer effect, and confirmed that the anti-cancer activity of BCG is derived from an inherent characteristics of DNA sequence. In 1995, Kreig, et al. proved, during the study of antisense oligonucleotides suppressing genes of a B cell, the fact that a synthetic oligodeoxynucleotides (ODNs) of a specific DNA sequence composed of unmethylated cytosine and guanine may induce activation of the immune cells. From the Kreig's aspect, it was newly presented that the anti-cancer effect of BCG proved by the Japanese researchers in the art is derived from the characteristics of unmethylated BCG DNA, and the immunological activation by such a bacterial DNA allows the immune system of the vertebrate to distinguish self DNA and non-self DNA.
The early studies of the immunological activation and its control by bacteria focused on protein antigens such as Coley's toxin, which induces generation of the antibody. However, many of the studies reported that more powerful inducers for the immunological activation are present among the components of the microorganism. And, it was also proved that the bacterial DNA is prone to induce a powerful immunological activation, and certain immune responses to each antigen (6, 7). An CpG dinucleotide composed of two nucleic acid sequences is a gist, of the immunological activation and its control, and it was revealed from the recent studies that the vertebrate also distinguishes self DNA from bacterial DNA to activate the immune cells. Such a CpG motif is plentiful in the bacteria, but not in the vertebrate. It was seen that an oligodeoxynucleotide including the CpG motifs (CpG-oligodeoxynucleotide, CpG-ODN) activates various defense mechanisms of the host including innate immune responses and acquired immune responses (Akdis, C A. Curr Opin Immunol., 12:641-646, 2000).
Recently, there has been developed a CpG-ODN whose backbone was modified so as to increase usability of the CpG-ODN. The CpG-ODN with a phosphodiester backbone referred to as a basic backbone of DNA, was easily decomposed in the body since it was sensitive to nucleases. Accordingly, the CpG-ODN has a low risk of inducing in vivo toxicity. However, it is revealed that the CpG-ODN with the phosphodiester backbone has a lower activity than the CpG-ODNs of the other backbones (Kwon, H J. et al., Biochem. Biophys. Res. Commun., 311:129-138, 2003). On the other hand, the CpG-ODN with the phosphorothioate backbone was artificially engineered by modifying its structure so that it cannot be decomposed in vivo by the nuclease. The CpG-ODN with the phosphorothioate backbone has a good in vivo stability and an excellent ability to activate the B cells, compared to the CpG-ODN with the phosphodiester backbone. Accordingly, the CpG-ODN modified into the phosphorothioate backbone has been widely used lately. However, such a CpG-ODN with the phosphorothioate backbone induces toxicity since it increase binding by the ODN non-specific to many proteins, and therefore it is not easily decomposed in vivo. Also, it was reported that the CpG-ODN with the phosphorothioate backbone induces the arthritis and exacerbates its symptoms (Deng G M et al., Arthritis & Rheumatism, 43 (2): 356-364, 2000), and causes the autoimmune-related diseases such as SLE (systemic lupus erythematosis) (Tanaka, T. et al., J Exp. Med. 175:597-607, 1992).
Formulations has been manufactured by adding various materials as the adjuvant to vaccine, and such a formulation has been designed to maximize an effect of the vaccine since the event of this century. However, aluminum salt (alum, Al2O3) is now only an adjuvant approved so that it can be administered in the vaccine. In the recent study, it was found that efficacy of the vaccine was much more excellent when a recombinant hepatitis surface antigen was mixed with the alum and the CpG-ODN and administered to a mouse than when only the alum was used as the adjuvant (Davis H L. et al. J. Immunol. 160: 870-876, 1998). It was seen that the alum slightly induces cell-mediated immunity by inducing the Th2 immune reaction, while the CpG ODN strongly induces humoral and cell-mediated immunity by inducing expression of the Th1 cytokines. However, the problem is that the CpG-ODN used in this case may cause a side effect since it has a phosphorothioate backbone.
Meanwhile, skin diseases are referred to as all abnormalities that appear in the skin of the animals including human. Amongst them, an atopic dermatitis has characteristic major symptoms such as chronic/inflammatory skin diseases selected from the group consisting of a serious pruritus, dry skins and an eczematous dermatitis (Rudikoff, D. et al. Lancet. 351:1715-1721, 1998). Generally, the atopic dermatitis tends to be inherited, and accompanied by an allergic asthma, an allergic rhinitis, an allergic conjunctivitis and an urticaria, depending on individuals. A series of immunological abnormalities reported in the atopic dermatitis patients include an increased production of IgE, the reduced number and deteriorated function of CD8+ suppressor/cytotoxic T lymphocytes, the reduced number of Th1 (T-cell Helper type 1) lymphocyte that secretes IFN-gamma, etc. Also, T lymphocyte having histological CD4+ phenotype, infiltration of monocytes/macrophages, mast cells and eosinophils are increased in the skin abnormality of the atopic dermatitis, and dendritic cells (DCs) and epidermal Langerhans cells are also increased in the skin abnormality of the atopic dermatitis (Imokawa, G., et al., J. Invest. Dermatol., 96:523-526, 1991).
Many researchers have developed the methods for treating the cancer by killing the cancer cells using X-ray. However, when the cancer is treated using the irradiation, cancer tissues and its adjacent immune cells all are inevitably damaged due to the irradiation, resulting in its reduced immune functions. It has been reported that the immune cells such as B cells (Ashwell J D et al., J. Immunol. 136:3649-3656, 1986), T cells (Prosser J S Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 30:459-465, 1976), macrophages (Yoshihisa K et al., J. Radiat Res. 45:205-211, 2004), etc. were killed by the irradiation (apoptosis). Accordingly, among the radiotherapeutic methods for treating the diseases such as a cancer, etc., there are required the methods that normal immune cells except the cancerous cells are survived to normally maintain the immune reactions.
The present invention relates to oligonucleotides derived from Mycobacterium bovis BCG for manipulating immune reactions, which may be used in treatment of various immune-related diseases by stimulating immune reactions (an adjuvant) and maintaining a balance of the immune reactions, and also have effects of treating an atopic dermatitis and increasing viability of the cells as a function of the irradiation.